Implantable prosthetic devices coated with bioactive molecules

ABSTRACT

Coated implantable prosthetic devices are disclosed. The device is a prosthetic having a gold layer on the surface to which bioactive molecules are attached through a gold-sulfhydryl bond. The devices are easy and convenient to prepare. Gold coated implantable devices are also disclosed herein. The gold coated implantable device is a prosthetic device formed of a porous non-fabric material having a surface with projections and indentations and the gold layer on the surface of the porous non-fabric material forms a uniform layer across the material such that the gold layer also forms projections and indentations.

BACKGROUND OF THE INVENTION

Implantable prosthetic devices have been used in the surgical repair orreplacement of internal tissue for many years. The efficacy of manytypes of implants is primarily dependent upon the surrounding tissue'sadaptive reformation around and ability to bond to the implant surface.In orthopedic implants in particular, the geometry and the quality ofbone reformation determines how much load the bone can resist.Orthopedic implants include a wide variety of devices, each suited tofulfill particular medical needs. Examples of such devices are hip jointreplacement devices, knee joint replacement devices, shoulder jointreplacement devices, and pins, braces and plates used to set fracturedbones. Some contemporary orthopedic implants, including hip and kneecomponents, use high performance metals such as cobalt-chrome andtitanium alloy to achieve high strength. These materials are readilyfabricated into the complex shapes typical of these devices using maturemetal working techniques including casting and machining.

At least two other methods are currently employed for bone and jointreplacement and repair. Those methods include: (1) the use of groutingmaterials such as poly(methyl methacrylate) (PMMA) as bone cementbetween the bone and the prosthesis; and (2) direct opposition of bonetissue onto porous and non-porous implant surfaces. The latter method isknown as the “cementless implant method.”

In one example of the cementless implant method, a prosthesis is coatedwith hydroxyapatite which is a major inorganic component of bone. Thehydroxyapatite-coated prosthesis is then implanted in the bone cavity.The hydroxyapatite, which is a calcium salt, is believed to facilitateosteointegration with the bone tissues. After partial integration of thehydroxyapatite-coated prosthesis with the bone, layers of hydroxyapatitecan be detected between the prosthesis and the bone tissues.

Despite the success of both metal and non-metal components in manypatients, long term data has demonstrated an unacceptably high failurerate in more active patients due to loosening of the implant caused bybone resorption around the implant or failure to achieve bone ingrowth.Bone resorption results from stress shielding of the bone around theimplant. The failure to achieve bone ingrowth into the surface of theimplant to support implant mechanical stability has been a major problemwith conventional implants. The metal orthopaedic prostheses rely onpoly(methyl methacrylate) for attachment and fixation to bone. Looseningof such implants as a result of cement failure has resulted inadditional surgeries for securing the implants. In order to avoid theproblems associated with these prostheses, prostheses having porous orcentered coatings have also been used. Although these materialsencourage tissue ingrowth, the process of ingrowth occurs over a periodof weeks to months, during which time the implant may be loosened andfail to function properly.

SUMMARY OF THE INVENTION

It has been discovered according to the present invention thatconventional implants can be improved by coating with a layer of goldand attaching to the gold a bioactive molecule. The bioactive moleculefunctions at the implant surface to promote a favorable, local,environmental response. Accordingly, the invention is an improvedimplantable prosthetic device coated with a bioactive molecule.

The prosthetic device provided according to the invention is convenientand simple to prepare. The bioactive molecules are directly coupled tothe prosthetic device surface through a gold-sulfide bond using simplesolution chemistry techniques. Prior art methods for modifying thesurface of biomaterials were complex and cumbersome. For instance, inorder to conjugate a molecule to a polymeric surface, the surface wouldfirst have to be modified to add a functional group to which themolecule could bind. In some cases the molecule would require theaddition of a linking group which is capable of reacting with thefunctional group.

According to one aspect, the invention is a prosthetic device includinga shaped substrate having a substrate surface, for implantation in amammal, a layer of gold attached to the substrate surface and defining atissue contacting surface, and a bioactive molecule bound to the goldlayer. The shaped substrate can be, for example, a polymer, a metal, aplastic, a fabric, a ceramic, a biological material, or a composite oftwo or more materials. The gold layer may be any thickness butpreferably the gold layer has a thickness of about 10 to 1000 Angstroms.The bioactive molecule in turn can form a monolayer on the surface ofthe gold which, depending on the size of the bioactive molecule, isabout 1 to 500 Angstroms in thickness.

The bioactive molecule can be virtually any molecule which can beattached to the gold layer and which can affect favorably the implant inits local environment once implanted. The bioactive molecule, therefore,can be natural or synthetic including a protein, a peptide, a proteinanalog, a sugar, a lipid, a glycol protein, a glycolipid or a nucleicacid. In one embodiment the bioactive molecule is selected from thegroup consisting of a cell modulating molecule, a chemotactic molecule,an anticoagulant moleucle, an antithrombotic molecule, an anti-tumormolecule, an anti-infectious molecule, a growth potentiating molecule,and an anti-inflammatory molecule. In one embodiment the cell modulatingmolecule is selected from the group consisting of an anti-integrinantibody, a bone morphogenic protein, an integrin binding protein, and acadherin binding protein. In another embodiment the chemotactic moleculeis an extracellular matrix molecule selected from the group consistingof collagen, fibronectin, laminin, and proetoglycan. In yet anotherembodiment the anti-tumor molecule is selected from the group consistingof methotrexate, adriamycin, cyclophosphamide, and taxol. Theanti-infectious molecule is selected from the group consisting ofantibiotics such as penicillin according to another embodiment. Inanother embodiment the growth potentiating molecule is selected from thegroup consisting of growth factors such as PDGF, EGF, FGF, TGF, NGF,CNTF, and GDNF. According to another embodiment the anti-inflammatorymolecule is selected from the group consisting of steroidal andnon-steroidal compounds.

The layer of gold can be attached directly to the substrate surface. Inanother embodiment the layer of gold is attached to the substratesurface via attachment to an intermediate layer, such as a layer oftitanium intermediate the gold layer and the substrate surface.

According to another embodiment the surface of the prosthetic device isformed of a porous material, wherein the layer of gold creates a goldsurface that has projections and indentations and wherein the layer ofgold has an approximately uniform thickness across the surface of theporous material.

According to another aspect, the invention is a prosthetic deviceincluding a shaped substrate having a substrate surface, forimplantation in a mammal, a layer of gold attached to the substratesurface and defining a tissue contacting surface, and a bioactivepeptide bound to the gold layer. The shaped substrate can be, forexample, a polymer, a metal, a plastic, a fabric, a ceramic, abiological material, or a composite of two or more materials. The goldlayer may be any thickness but preferably the gold layer has a thicknessof about 10 to 1000 Angstroms. The bioactive peptide forms a monolayeron the surface of the gold which, depending on the size of the peptide,is about 1 to 500 Angstroms in thickness.

The bioactive peptide can be any peptide which can be attached to thegold layer and which can affect favorably the implant in its localenvironment. It can be natural or synthetic. In one embodiment thebioactive peptide is selected from the group consisting of a cellmodulating peptide, a chemotactic peptide, an anticoagulant peptide, anantithrombotic peptide, an anti-tumor peptide, an anti-infectiouspeptide, a growth potentiating peptide, and an anti-inflammatorypeptide. In one embodiment the cell modulating peptide is selected fromthe group consisting of an anti-integrin antibody fragment, a cadherinbinding peptide, a bone morphogenic protein fragment, and an integrinbinding peptide. Preferably the cell modulating peptide is a integrinbinding peptide which is selected from the group consisting of RGDC,RGEC, RGDT, DGEA, DGEAGC, EPRGDNYR, RGDS, EILDV, REDV, YIGSR, SIKVAV,RGD, RGDV, HRNRKGV, KKGHV, XPQPNPSPASPVVVGGGASLPEFXY, andASPVVVGGGASLPEFX. The peptides also may be any functionally activefragment of the proteins disclosed herein as being bioactive moleculesuseful according to the invention. In another embodiment the chemotacticpeptide is selected from the group consisting of functionally activefragments of collagen, fibronectin, laminin, and proteoglycan. In yetanother embodiment the anti-tumor peptide is selected from the groupconsisting of functionally active fragments of protein anti-tumoragents. The anti-infectious peptide is selected from the groupconsisting of functionally active fragments of the proteinanti-infectious agents according to another embodiment. In anotherembodiment the growth potentiating peptide is selected from the groupconsisting of functionally active fragments of PDGF, EGF, FGF, TGF, NGF,CNTF, GDNF, and type I collagen related peptides. According to anotherembodiment the anti-inflammatory peptide is selected from the groupconsisting of functionally active fragments of anti-inflammatory agents.

The layer of gold can be attached directly to the substrate surface. Inanother embodiment the layer of gold is attached to the substratesurface via attachment to an intermediate layer, such as a layer oftitanium intermediate the gold layer and the substrate surface.

According to another embodiment the surface of the prosthetic device isformed of a porous material, wherein the layer of gold creates a goldsurface that has projections and indentations and wherein the layer ofgold has an approximately uniform thickness across the surface of theporous material.

The invention in another aspect is a prosthetic device including ashaped substrate formed of a textured material having a substratesurface with first projections and first indentations and a layer ofgold attached to the substrate surface of the textured material, whereinthe layer of gold creates a gold surface that has second projections andsecond indentations corresponding to the first projections and firstindentations. In one embodiment, the layer of gold has an approximatelyuniform thickness across the substrate surface of the textured material.Preferably the textured material is a porous material such as a poroustitanium material, a porous polymer, or any other non-fabric porousmaterial.

In one embodiment the textured material is a polymer. In anotherembodiment the gold layer has a thickness of about 10 to 1000 Angstroms.

According to yet another embodiment the prosthetic device also includesa layer of bioactive peptide attached to the gold surface through agold-sulfide bond.

In another aspect the invention is a prosthetic device including ashaped substrate having a substrate surface, a layer of gold attached tothe substrate surface, and an RGDC peptide attached to the gold layerthrough a gold-sulfide bond. According to an embodiment the shapedsubstrate is a polymer, a metal, a plastic, a fabric, a ceramic, abiological material, or a composite of two or more materials. In oneembodiment the gold layer has a thickness of about 10 to 1000 Angstroms.In another embodiment the bioactive peptide forms a layer about 1 to 500Angstroms in thickness.

The layer of gold is attached directly to the substrate surface in oneembodiment. In another embodiment the layer of gold is attached to thesubstrate surface via attachment to a layer of titanium intermediate thegold layer and the substrate surface.

According to another embodiment the surface of the prosthetic device isformed of a porous material, wherein the layer of gold creates a goldsurface that has projections and indentations. In one embodiment thelayer of gold has an approximately uniform thickness across the surfaceof the porous material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the observed reflectivity change uponincubation of a clean gold surface with a 0.2 mM solution of the RGDCpeptide;

FIG. 2 is a graph depicting the SPR spectra taken in an air ambientbefore and after adsorption of the RGDC peptide layer; and

FIG. 3 is a graph depicting alkaline phosphatase activity fromosteoblasts cultured on RGDC-gold coated, CGRARADSP-gold coated, andplain gold surfaces.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, it was discovered that animplantable device could be coated with a bioactive molecule by firstcoating a substrate with a gold layer and then attaching the bioactivemolecule through a simple reaction to the gold layer by forming agold-sulfide bond. Prior art methods for attaching molecules to thesurface of materials are cumbersome. In order to make a polymeric orother non-metal prosthetic device coated with molecules using theseprior art methods the surface of the prosthetic device would have to bemodified and would most likely require the addition of couplingreagents, making the preparation of such devices expensive, timeconsuming, and impractical. The preparation of metal implants havingmolecules attached to surfaces of the implants has been a difficultchallenge in the prior art because most metal surfaces have oxide layerswhich make binding of coupling agents difficult. The implantableprosthetic device coated with bioactive molecules disclosed herein isprepared by a simple technique for coupling bioactive molecules tobiomaterial surfaces.

The function performed by the surface is defined in part by the type ofbioactive molecule bound to the surface. As used herein a “bioactivemolecule” is any biologically active molecule which includes asulfhydryl group or to which a sulfhydryl group can be attached directlyor indirectly. Examples are a peptide, protein (e.g., apoprotein,glycoprotein, antigen and antibody), a protein analog containing atleast one non-peptide linkage in place of a peptide linkage, a nucleicacid, etc. Nucleic acids include nucleotides; oligonucleotides; andtheir art-recognized and biologically functional analogs and derivativesincluding, for example, oligonucleotide analogs having phosphorothioatelinkages.

Preferred bioactive molecules include a cell modulating molecule, achemotactic molecule, anticoagulant moleucle, antithrombotic molecule,an anti-tumor molecule, an anti-infectious molecule, a growthpotentiating molecule, and an anti-inflammatory molecule.

A cell modulating molecule as used herein is a molecule that interactswith a cell and modifies the cell in any way e.g. alters geneexpression, such as bone morphogenic protein, anti-integrin antibodies,integrin binding protein, and cadherin binding protein.

A chemotactic molecule as used herein is a molecule which attracts cellsto a surface or aids in a cell's attachment to a surface and includesextracellular matrix proteins such as collagen, fibronectin, laminin,and proetoglycan.

An anti-tumor molecule as used herein is a molecule which decreases orprevents a further increase in growth of a tumor and includesanti-cancer agents such as Acivicin; Aclarubicin; AcodazoleHydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-Ib;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride, and Taxol.

An anti-infectious molecule as used herein is a molecule which reducesthe activity of or kills a microorganism and includes Aztreonam;Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; PirazmonamSodium; Propionic Acid; Pyrithione Sodium; Sanguinarium Chloride;Tigemonam Dicholine; Acedapsone; Acetosulfone Sodium; Alamecin;Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin;Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefmenoxime Hydrochloride;Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; CefotetanDisodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol PantothenateComplex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline HydrochlorideTetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin;Zorbamycin; Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide;Moxalactam Disodium; Ornidazole; Pentisomicin; and SarafloxacinHydrochloride.

A growth potentiating molecule as used herein is a molecule whichstimulates growth of a cell and includes growth factors such as PDGF,EGF, FGF, TGF, NGF, CNTF, and GDNF.

An anti-inflammatory molecule as used herein is a molecule which reducesan inflammatory response and includes steroidal and non-steroidalcompounds; Alclofenac; Alclometasone Dipropionate; Algestone Acetonide;Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; AmipriloseHydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; BalsalazideDisodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; s Flunisolide Acetate; Flunixin; Flunixin Meglumine;Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen;Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide;Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen;Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin;Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; IsoflupredoneAcetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride;Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium; MeclofenamicAcid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

An anticoagulant moleucle as used herien is a molceule that preventsclotting of blood and includes but is not limited to Ancrod;Anticoagulant Citrate Dextrose Solution; Anticoagulant Citrate PhosphateDextrose Adenine Solution; Anticoagulant Citrate Phosphate DextroseSolution; Anticoagulant Heparin Solution; Anticoagulant Sodium CitrateSolution; Ardeparin Sodium; Bivalirudin; Bromindione; Dalteparin Sodium;Desirudin; Dicumarol; Heparin Calcium; Heparin Sodium; Lyapolate Sodium;Nafamostat Mesylate; Phenprocoumon; Tinzaparin Sodium; Warfarin Sodium.

An antithrombotic moleucle as used herien is a molceule that preventsformation of a thrombus and includes but is not limited to AnagrelideHydrochloride; Bivalirudin; Dalteparin Sodium; Danaparoid Sodium;Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium;Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; Trifenagrel.

Preferably the bioactive molecule is a bioactive peptide. A “bioactivepeptide” as used herein refers to oligopeptides having a chain of lessthan or equal to fifty amino acids and which is capable of performing adesired biological function. In a preferred embodiment the bioactivemolecule includes a cell modulating peptide, a chemotactic peptide, ananticoagulant peptide, an antithrombotic peptide, an anti-tumor peptide,an anti-infectious peptide, a growth potentiating peptide, and ananti-inflammatory peptide. A cell modulating peptide includes, forexample, an antibody fragment or an integrin binding peptide. Bioactivepeptides include peptide fragments of the proteins which are bioactivemolecules disclosed herein and having the functional properties of thoseproteins.

A preferred use for the peptide-coated implantable device of theinvention is for enhancing and/or accelerating bone growth in areas ofdamaged bone or in bone replacement surgery. Bone and joint replacementsurgeries are commonly used, for instance, to relieve pain, improvefunction, and enhance the quality of life for patients with medicalconditions caused by osteoarthritis, rheumatoid arthritis,post-traumatic degeneration, avascular necrosis, and other aging-relatedconditions. The prosthetic device of the invention which is coated withbioactive peptides that enhance or accelerate bone growth significantlyimprove the ability of an implant to remain attached to the bonesurface. Preferred integrin binding peptides which perform this functionare RGDC, RGEC, RGDT, DGEA, DGEAGC, EPRGDNYR, RGDS, EILDV, REDV, YIGSR,SIKVAV, RGD, RGDV, and HRNRKGV.

Anti-infectious peptides include include antibiotic peptides such asthose disclosed in U.S. Pat. No. 5,602,097. Anti-tumor andanti-infectious peptides are also disclosed in U.S. Pat. No. 5,516,755.U.S. Pat. No. 5,484,885 discloses chemotactic, antibiotic, andlipopolysaccharide binding peptide fragments of CAP37 protein. Thesepeptide sequences are approximately five consecutive amino acids long.U.S. Pat. No. 5,354,736 discloses several collagen type I relatedpeptides which are useful for promoting growth.

Growth potentiating peptides also include low molecular weight tibialgrowth potentiating peptides such as those disclosed in U.S. Pat. No.5,576,301. These peptides are useful for potentiating tibial growth.These peptides have the following sequences: XPQPNPSPASPVVVGGGASLPEFXYand ASPVVVGGGASLPEFX.

Bioactive peptides such as those disclosed above are well known in theart. Other bioactive peptides useful according to the invention may beidentified through the use of synthetic peptide combinatorial librariessuch as those disclosed in Houghton et al., Biotechniques, 13(3):412-421(1992) and Houghton et al., Nature, 354:84-86 (1991) or using phagedisplay procedures such as those described in Hart, et al., J. Biol.Chem. 269:12468 (1994). Hart et al. report a filamentous phage displaylibrary for identifying novel peptide ligands for mammalian cellreceptors. In general, phage display libraries using, e.g., M13 or fdphage, are prepared using conventional procedures such as thosedescribed in the foregoing reference. The libraries display insertscontaining from 4 to 80 amino acid residues. The inserts optionallyrepresent a completely degenerate or a biased array of peptides. Ligandsthat bind selectively to a specific molecule such as a cell surfacereceptor are obtained by selecting those phages which express on theirsurface a ligand that binds to the specific molecule. Ligands thatpossess a desired biological activity can be screened in knownbiological activity assays and selected on that basis. These phages thenare subjected to several cycles of reselection to identify thepeptide-expressing phages that have the most useful characteristics.Typically, phages that exhibit the binding characteristics (e.g.,highest binding affinity or cell stimulatory activity) are furthercharacterized by nucleic acid analysis to identify the particular aminoacid sequences of the peptides expressed on the phage surface and theoptimum length of the expressed peptide to achieve optimum biologicalactivity. Alternatively, such peptides can be selected fromcombinatorial libraries of peptides containing one or more amino acids.Such libraries can further be synthesized which contain non-peptidesynthetic moieties which are less subject to enzymatic degradationcompared to their naturally-occurring counterparts. U.S. Pat. No.5,591,646 discloses methods and apparatuses for biomolecular librarieswhich are useful for screening and identifying bioactive peptides.Methods for screening peptides libraries are also disclosed in U.S. Pat.No. 5,565,325.

Peptides obtained from combinatorial libraries or other sources can bescreened for functional activity by methods known in the art. Forinstance when the peptide is a cell modulating peptide, and inparticular an integrin binding peptide, one of ordinary skill in the artcan easily determine whether the peptide will modulate bone cellactivity by performing the in vitro studies set forth in example 2 tomeasure osteoblast differentiation. Likewise, similar experiments can beconducted for other types of cells using cell specific markers ofdifferentiation or growth. The type of assay of course, used for aparticular peptide depends on the source of the peptide. For instance ifa peptide is a fragment of an anti-tumor molecule, the peptide should betested for functional activity in an anti-tumor assay. Those of skill inthe art can easily choose an appropriate assay for testing functionalityof a particular peptide.

The bioactive molecules useful according to the invention arecommercially available from many sources and methods for making thesemolecules also are well known in the art. Bioactive peptides andproteins may easily be synthesized or produced by recombinant means.Such methods are well known to those of ordinary skill in the art.Peptides and proteins can be synthesized for example, using automatedpeptide synthesizers which are commercially available. Alternatively thepeptides and proteins can be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

The bioactive molecule is bound to a gold surface. Although manyattempts have been made in the prior art to coat peptides, proteins andother biomaterials on various surfaces, each of these techniques hasrequired the use of complex coupling techniques and surface modificationincluding the use of coupling agents and linkers. It has been discoveredaccording to the present invention that bioactive molecules can beattached to a prosthetic device via a gold surface, through a simpletechnique that results in the formation of a bond between a gold and asulfhydryl group. The bond that forms between a sulfhydryl group andgold only requires the interaction between the sulfhydryl group and thegold in a solution. The interaction does not require coupling agents orlinkers or surface activation or modification of the gold.

The molecule is added to the gold surface using simple solutionchemistry techniques, e.g., simply exposing the gold surface to asolution of molecule in a solvent such as ethanol:water. This approachis simple and is non-line of sight dependent. A technique which is lineof sight dependent only coats an external surface and does not coatinternal pores or interstices. Non-line of sight dependent methods arecapable of coating the internal surface area such as pores. Thistechnique produces an evenly coated layer of molecule on any type ofdevice, even those having a porous, spongy, or textured surface.

Bioactive molecules can be attached to gold surfaces directly or viaspacers. If direct, then bioactive molecules must have (or must bemodified to have) a sulfhydryl group. If indirect, the bioactivemolecule may or may not have sulfhydryl, but the spacer will have asulfhydryl. In this instance the spacer is attached to the gold surfaceand the bioactive molecule is attached to the spacer, before or afterattaching of the spacer to the gold surface. Proteins or peptides havingendogenous cysteine groups already have a cysteine within the moleculeand do not require the addition of another sulfhydryl group. If aprotein or peptide has more than one cysteine and those cysteines haveformed di-sulfide bridges the molecule can be subjected to reducingagents to ensure that the sulfhydryl group is free and available.

Proteins or peptides without endogenous cysteine groups can easily bemanipulated to incorporate a sulfhydryl group. For instance, peptidesand proteins can be subjected to site directed mutagenesis to prepare acysteine containing protein or peptide. Additionally a cysteine can beadded to either the N-terminal or C-terminal of the peptide or proteinor incorporated within the peptide or protein or within a branch of thepeptide or protein. A cysteine may be added anywhere in the peptide orprotein that does not affect the biological activity of the peptide orprotein. This is demonstrated schematically as follows:

wherein X, Y, and Z are any amino acid and C is cysteine. Preferably acysteine group is added to either the C-terminal or the N-terminal ofthe peptide. More preferably, the cysteine group is on the C terminalregion of the peptide.

Proteins or peptides without endogenous cysteine and othernon-sulfhydryl containing molecules can easily be manipulated toincorporate a non-cysteine sulfhydryl group. For example, sulfhydrylgroups can be introduced into the molecules having a primary amine (ormodified to have a primary amine) by reaction of the primary amine inthe molecule with 2-iminothiolanc or Traut's reagent, or othercommercially available reagents. A variety of commercially availablereagents for coupling sulfhydryl groups to molecules are available fromPierce Chemical, Corp., such as Traut's reagent (Product No. 26101),SATA (Product No. 26102) or SPDP (Products Nos. 21757, 21657, 21557).Traut's reagent is a water soluble reagent which reacts with primaryamines at pH 7-10 to introduce sulfhydryl groups, as disclosed in Schramand Dulffer, Physiol. Chem., 358, 137-139 (1977). Traut's reagent hasthe following structure:

SATA is a reagent which adds protected sulfhydryls to molecules byreacting with primary amines. SATA has the following chemical structure:

SPDP, which includes LC-SPDP and Sulfo-LC-SPDP also is capable of addinga sulfhydryl group to primary amines. These molecules have the followingstructures:

Preferably, the bioactive molecule is prepared with a sulfhydryl groupat, for example, the carboxyl (C) or amino (N) terminus and then iscoupled to the gold surface. In an alternative embodiment, a spacer issynthesized with a sulfhydryl group, preferably at or near one end, andthen this spacer is attached at this end to the gold surface and via adifferent functional group to the bioactive molecule. The spacermolecule may be coupled for example to the terminal amine group orcarboxyl group of the bioactive peptide or protein. Spacer molecules canbe selected, for example, which contain (or which can be modified tocontain) a functional group that is reactive with the peptide or proteinN-terminal amine group and allowing the functional group and the peptideor protein N-terminal amine to form a linkage in accordance withart-recognized procedures. See, e.g., March, J., Advanced OrganicChemistry, 4th Ed., New York, N.Y., Wiley and Sons, 1985), pp.326-1120.In an analogous manner, the spacer molecule may be coupled to a reactivegroup in the C-terminus of the bioactive peptide or protein.Additionally the spacer molecule may be coupled to a branch of amolecule or an internally active portion of a molecule or any end group.

Thiol or amide groups may be added at any nucleotide of a nucleic acid.The amine group may be added so as to provide a point of attachment fora sulfhydryl group by the above-described reagents. Nucleic acids mayalso be synthesized with groups such as amine groups.

The bioactive molecule is bound to a layer of gold which is attached toa substrate surface of a shaped substrate. The layer of gold covers allor part of the prosthetic device to define a tissue contacting surface.The tissue contacting surface is the surface of the gold to which themolecules are bound. The layer of gold may be extremely thin or it maybe thick. The layer of gold may actually be the entire prostheticdevice. In this case the layer of gold would encompass the shapedsubstrate as well. Preferably the layer of gold is thin because of thehigh cost of gold.

The layer of gold is attached to the shaped substrate surface by anymeans known in the art. For instance, the gold layer can be added to theimplant using evaporation, electroplating, sputtering orelectrodeposition. Using any of these techniques the gold can be appliedin a thin layer to the surface of the implant. Preferably the gold isattached to the substrate by electroplating or evaporation.Electroplating produces a gold layer which is non-line of sitedependent. Using electroplating, therefore, a gold layer can be producedon an uneven surface such that the uneven nature of the surface ismaintained.

A shaped substrate as used herein is a material which has the shape ofan implantable prosthetic. The selection of the shape of the prostheticis governed by the physical requirements of space, geometry and functionat the region where the implant is to be positioned in the body.Implants can be made available in a range of sizes to fit the varyingsizes in the patient population.

In some embodiments, the bioactive molecule coating is on and within thepores of an implantable prosthesis of the type where tissue ingrowth iscontemplated, wherein the bioactive molecule encourages the ingrowth ofthe tissue into the pores or facilitates attachment of tissue to theprosthetic. In another embodiment, the coating is on a typicalprosthesis or on a ‘temporary implant’, such as a long term buttemporary catheter, and the coating is of an antibacterial agent toprevent colonization upon the prosthesis or catheter. Thus, theinvention is useful in connection with prosthetic devices such as boneor joint replacement or repair prosthetics, vascular prostheses,including woven prostheses, catheters for implantation and the like.Virtually any implantable tissue contacting surface may be modified asdescribed herein.

The shaped substrate may be made from any material ordinarily used toprepare implants. For instance the shaped substrate may be made from anyof a wide variety of metals, such as, pure titanium, titanium alloy,stainless steel, cobalt-chrome alloy, and gold. The shaped substrate mayalso be made from polymeric matrix composites, such as continuousfilament carbon, graphite, glass and aramid fibers embedded within apolymer matrix, such as polysulfone, polyether-ether-ketone,polyether-ketone-ketone, polyimide, epoxy or polycyanate, polymersincluding polyethylene, polyetheretherketone (PEEK), polypropylene,polymethylmethacrylate, polyamides, and polyester. Other polymericmatrix composites include but are not limited to polyethylene films,ultra-high molecular weight polyethylene films and fibers,polyvinylidene fluoride films, poly(methyl methacrylate) films,polystyrene films, nylon 12 films and fibers, various polyesters andpolyacrylates, polyetherethereketones, aromatic polyamides, polyethyleneterephthalate fibers and films, poly(tetramethylene terephthalate)films, and polyether-esters of poly(tetramethylene terephthalate).

The prosthetic device of the invention is useful for implantation inmammals. Mammals herein means humans, cats, dogs, mice, hamsters, pigs,goats, primates, horses, cows, and sheep.

A preferred prosthetic device of the invention is a shaped substratehaving a substrate surface, a layer of gold attached to the substratesurface, and an RGDC peptide attached to the gold layer through agold-sulfide bond. The RGD peptide is a peptide found in manyextracellular matrix proteins which is known to bind α₅β₁ and α_(v)β₃integrin receptors. RGD attached to surfaces has been demonstrated toincrease osteoblast attachment to the surface. It is preferred thatorthopedic prosthetic devices are coated with RGDC.

The prosthetic device with the bioactive molecule attached to thesurface has been found to be extremely stable and as a result can bestored for extended periods of time. The stability of the device isimportant because it enables the device to be prepared in advance andshipped to a medical institution where it can be stored for futureimplantation. As a result medical institutions can store many prostheticdevices having various molecules already coated on the surface forvarious applications.

The prosthetic device of the invention may also be prepared and storedwithout the bioactive molecule attached to the device. The bioactivemolecule can then be added at a later time point prior to use. The stepof adding the bioactive molecule to the gold surface is simple and quickand may easily be performed immediately prior to a surgical process.Accordingly, the prosthetic device of the invention also includes ashaped substrate formed of a textured material and having a gold layerattached to the surface. More specifically the shaped substrate has asubstrate surface with first projections and first indentations and alayer of gold is attached to the substrate surface of the texturedmaterial such that the layer of gold creates a gold surface that hassecond projections and second indentations corresponding to the firstprojections and indentations. The layer of gold optionally has anapproximately uniform thickness across the substrate surface of thetextured material.

A “textured material” as used herein is a non-fabric material havingsmall (about 1-1000 microns in size) interstices throughout. The shapedsubstrate may be made entirely of a textured material or may optionallybe made of a non-textured material but having a surface which is coatedwith a textured material to produce a textured surface. Preferably thetextured material is a porous material such as a porous titaniummaterial, a porous polymer, or any other non-fabric porous material.Porous metal surfaces have been created by plasma spraying (U.S. Pat.No. 3,605,123) of fine metallic particles, or by sintering a looselypacked coating of metallic particles (U.S. Pat. No. 4,550,448, BritishPatent No. 1,316,809), or by diffusion bonding kinked fiber metal pads(U.S. Pat. No. 3,906,550). Plasma spraying employs super heated gases tomelt the metal particles to be sprayed. Sintering develops interparticlebonds in a porous coating by exposing the coating and implant metal totemperatures approaching their melting point, while diffusion bondingemploys heat and pressure to promote atomic diffusion at the coatingimplant interface. Methods for preparing porous polymer materials arewell known in the art. Additionally the shaped substrate may be made ofa non-textured material but having a surface which is at least partiallycoated with a textured material to produce a partially textured surface.Thus the invention also encompasses a prosthetic device having a shapedsubstrate made from a non-textured material but at least partiallycoated with a textured material on which a layer of gold is attached.

The substrate surface of the textured material has projections andindentations. “Projections and indentations” as used herein aremicroscopic cavities on the surface of the substrate defining a ‘rough’surface microscopically. A substrate surface is said to have projectionsand indentations if it has a substantial region that is mostly free of aflat smooth surface, but instead is characterized by numerousindentations and projections throughout the region, numerous cavitieshaving a diameter between 1 micron and 1 millimeter, preferably between20 microns and 900 microns. In a preferred embodiment the gold layerattached to the textured material creates a gold surface that also hasprojections and indentations and that has an approximately uniformthickness across the substrate surface.

The following examples are provided to illustrate the methods andproducts of the present invention. As described above, many variationson these particular examples are possible and, therefore, the examplesare merely illustrative and not limiting of the present invention. Asdemonstrated in the Examples below the implantable prosthetic device ofthe invention has many advantages over uncoated implants and even overpeptide-coated implants that do not have a gold surface.

EXAMPLES

The following examples describe experiments which were conducted onmolecule-coated gold surfaces. Experiments were also carried out onmolecule coated polymer surfaces (FEP) which serve as controls. Bycomparing the results obtained with the gold-coated substrate with thoseobtained with the FEP material it is possible to distinguish the effectswhich occur as a result of the immobilized peptide from those whichoccur as a result of the surface context of the immobilized peptide.

Example 1 Immobilization of Peptides on Biomaterial Surfaces

Methods and Materials

Peptides: The peptides used in the following studies are set forth inTable I. All peptides were synthesized commercially (QCB, Hopkinton,Mass.) to a purity of 98% or greater by HPLC and mass spectrometry.Peptides being coupled to FEP (the control substrate) included G or GGGGspacer sequence on their N- or C-terminus. Peptides being coupled togold coated surfaces included a CG or CGGG spacer sequence on their N-or C-terminus. Control peptides were fabricated using scrambledsequences or, if known, amino acid substitutions.

TABLE 1 Extracellular Matrix Protein Peptide Ligand Integrin ReceptorsCollagen I cRGD, RGDT, DGEA α₁β₁, α₂β₁, α₃β₁ Bone Sialoprotein EPRGDNYRα_(v)β₃ Osteopontin RGD α_(v)β₃ Fibronectin RGDS, EILDV, REDV α₃β₁,α₄β₁, α₅β₁, α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α₄β₇ laminin YIGSR,SIKVAV, RGD α₁β₁, α₂β₁, α₃β₁, α₆β₁, α₇β₁, α₆β₄ Thrombospondin RGDα_(v)β₃ Vitronectin RGDV, HRNRKGV α_(v)β₁, α_(v)β₃, α_(v)β₅ OsteonectinKKGHK ? (SPARC)

Human and rat osteoblast/osteocytes express a range of integrins. Theseare shown in Table 2 below.

TABLE 2 Rat Calvarial Osteoblasts α₁β₁, α₅β₁, α_(v)β₁, α_(v)β₃, α_(v)β₅Human Osteoblasts α₃β₁, α₄β₁, α₅β₁, α_(v)β₃

Preparation of Gold Coated Substrates

12-mm diameter pre-cleaned circular glass cover slips were obtained fromFisher Scientific and placed into a custom mount consisting of a 0.25inch thick aluminum plate. The samples were then suspended inside afour-source NRC 3177 electron beam evaporator with a Sloan 180° electrongun and Sloan Six/Ten power supply. The gold coating did not adhere wellto plain glass, so titanium was used as an intermediate. The evaporatorchamber was pumped down to achieve a vacuum in the low 10⁻⁶ to high 10⁻⁷torr range. Initial pumping was done with a mechanical pump and then adiffusion pump was brought on line to achieve and maintain the finalpressure. A liquid nitrogen trap was employed to keep the system free ofcontaminating vapors from diffusion pump oil or other contaminants. Theelectron beam gun was activated and a 60 angstrom coating of Ti was putonto the cover slips. The Ti source was then rotated away as the goldsource was rotated into place. A 500 angstrom layer of gold was applied.The samples were then removed from the system and kept under nitrogen orcovered in Kimwipes and aluminum foil until ready for use.

Immobilization of peptides on Gold Coated Substrates

Cysteine terminated peptides were solubilized in a 1:1 ethanol:distilledwater solution at a concentration of 0.22 mM. The gold substrates wereexposed to this solution for one hour. Plain gold controls were made byexposing samples to peptide-free ethanol:distilled water for one hour.Reactions were carried out in the dark to protect the light-sensitivecysteine.

Preparation of FEP Membranes with Immobilized Peptide

FEP membranes with immobilized peptide are useful for comparisonpurposes. The FEP membranes were prepared using surface modification andcoupling techniques. FEP films (Dupont) 25 micrometers thick were cutinto discs with a lathe (1.76 cm diameters) and cleaned by sonication inhexanes and methanol for 20 seconds each. Surface hydroxyl (OH) groupswere added to cleaned FEP films by a radio frequency glow discharge(RFGD) process. The films were placed in a chamber and brought to apressure of 100 millitorr. The chamber was filled with hydrogen andmethanol vapor at 500 millitorr for 10 minutes. The pressure was againreduced to 100 millitorr and the radio frequency glow discharge wasactivated for 1 minute.

After rinsing the hydroxylated REP films in DMSO, the films were reactedwith CDI (40 mg/1 ml in DMSO) for 24 hours. To enhance nucleophilicattack of the OH— group to the CDI substrate, the solution wassupplemented with N-Hydroxy-succinimide (NHS, Fluka) (1 mg/ml in DMSO)(Frost, 1981) The excess CDI/NHS was rinsed off of the films with DMSObefore applying the peptide solution. Films were placed in 0.22 Mpeptide in 1M MES buffer (pH 5) for 48 hours (Hearn, 1987). Films wererinsed sequentially with 1M MES buffer, 1M NaCl, and distilled water.This stringent rinsing protocol was used to remove adsorbed vs. linkedpeptide from the surface.

The chemical reactions for the CDI Activation and the peptide couplingreactions are as follows:

The scheme for FEP and gold coated substrates are shown below.

Methods for Surface Characterization

1. Contact Angle

Contact angles were measured with ethylene glycol, glycerol, distilledwater, and ethanol on a goniometer. Each fluid was placed on thesubstrate using a syringe with a 30 gauge needle. At least threemeasurements per drop were taken. The surface energy was calculatedusing E. Sacher's method (Ratner, 1988; Kaelble, 1974; Kaelble, 1970).Contact angle data provides information regarding the surface chemistryand surface energetics of the top 5 Angstroms of a polymer substrate. Abead of pure liquid with a known surface tension is placed on thepolymer surface. The resulting bead angle is measured using a goniometer(an alternate technique is to use a Cahn microbalance). A hydrophobicsurface causes liquid beading and a high contact angle while a morehydrophilic surface is wettable and a small contact angle is observed. Arange of fluids with polar (i.e. water) to non-polar (i.e. decane)characteristics are tested.

2. Surface-Plasmon Resonance

Surface plasmon resonance (SPR) was produced when a beam of p-polarizedlaser light impinges onto the surface of a thin metal film. The lightwas coupled to the metal film through a prism which was mounted on arotating turntable. At a particular angle of incidence the E-field ofthe laser light interacts with the surface bound free electrons of themetal film in such a way that a charge density wave was generated at theinterface of the metal and air. This excitation results in a sharpreduction in the magnitude of the reflected light (measured with aphotodiode). The angle at which this occurs together with the depth andhalf-width of the minimum were determined by the thickness and complexrefractive index of the metal film. The magnitude of the evanescentfield which arises from the charge density wave decays exponentially inthe direction normal to the surface. Consequently, any dielectric layer(such as a peptide overlayer or cell membrane) adhering to the metalfilm will cause a change in the condition for resonance. By fitting theFresnel equations, firstly to the date for the uncoated metal film, andthen to the metal plus thin film, the thicknesses and complex refractiveindices of the metal and peptide overlayer were determined. Typicalthickness resolution for the SPR were of the order of 0.01 nm making itan extremely sensitive probe for the surface chemistry of peptides andproteins. By observing changes in reflectivity at a fixed angle ofincidence, it is possible to monitor the adsorption of peptides fromsolution onto a surface and thus obtain time resolved binding ofmolecules from the bulk to a surface. Another practical advantage ofthis method is that peptide chemistry can be determined in aqueousenvironments rather than the ultrahigh vacuums needed for othersurface-sensitive techniques (e.g. ESCA).

3. Characterization of Peptide Stability

a. Fluorescent Tagging of Immobilized Peptides

5-(and-6)-carboxythtramethylrhodamine succinimidyl ester, i.e. TAMRA SE(Molecular Probes #C-1171), reacts preferentially with amines. TAMRA SEhas the advantage of maintaining stability for weeks and is stable inpH's ranging from 4 to 9. The excitation and emission wavelengths ofthis compound are 546λ and 576λ, respectively. TAMRA SE is made up as a1 mM solution in DMF. It is then mixed with a pH 8.5 sodium tetraboratebuffer in a 1:1 ration for a final solution concentration of 0.5 mM.This is reacted with the samples on a stirrer plate for four hours.Rinsing is done overnight in 4 M urea+0.6% Tween 60.

b. Analysis of Peptide Stability under Physiologic Conditions

Various immobilized peptides, tagged with fluorescent probes, areexposed to tissue culture media, tissue culture media with 10% serum,and osteoblasts. After 1, 7, 14 and 28 days of culture, substrates arerinsed several times, and assessed for fluorescence using confocalmicroscopy.

Results

1. Contact Angle

Contact angles on pure gold are greatly effected by hydrocarbonsadsorbed from air, but surface energy was performed on thesecontaminated surfaces anyway as any implants are likely to be maintainedin air and are all going to be thus contaminated. Contact angles weremeasured with ethyleneglycol, glycerol, distilled water and ethanol on agoniometer. Each fluid was placed on the substrate using a syringe witha 26 gauge needle. The smallest drop possible was used to minimizegravitational effects. At least three measurements per liquid-samplecombination were taken. The surface energy was calculated using Sacher'smethod. The results gave us the following surface energies:

Material Surface Energy Plain Gold: 27.4 dyne/cm Gold + RGD: 25.0dyne/cm Gold + CG: 81.9 dyne/cm Gold + RGDC: 42.1 dyne/cm

On unmodified FEP, water generated a contact angle of approximately 105°indicating an unwettable, low energy surface. RFGD treated FEP showed acontact angle of 60-65° with water confirming the presence of polarhydroxyl groups.

2. Surface Plasmon Resonance

The incubation of a pure gold surface with a 0.22 mM solution of theRGDC peptide results in rapid film formation. Presumably the rapidadsorption is driven by the strong interaction of the cysteine residueof the peptide with the gold surface. The data is shown in FIG. 1.

FIG. 1 is a graph of the observed reflectivity change upon incubation ofa clean gold surface. The spectra were fitted using fresnel reflectivitytheory. Fitting the bare substrate spectra yielded optical constants forthe gold film of: Refractive index (n,k)=0.26708, −3.304, Film thicknessof 482 Å, Fit error=8.4×10⁻³. Using these constants for the gold film,the SPR spectra of the RGDC layer was analyzed to obtain the thicknessand refractive index of the peptide layer. The refractive index andthickness of the RGDC layer were allowed to vary between sensible limitsduring the fitting procedure, the best fit to the data yielded thefollowing parameters for the RGDC layer: Refractive index (n,k)=1.4665,−0.0992, Film thickness of 23-25 Å, Fit error=4.36×10⁻³.

FIG. 2 depicts a theoretical curve for the RDGC layer which wasgenerated using the above parameters. The film thickness value of 23-25Å indicates that the peptide molecules are in an upright orientation.

Non-SH containing RGD failed to bind to the gold surface.

Example 2 Evaluation of the Effects of Immobilized Peptides onOsteoblast Differentiation In Vitro

Rat calvarial osteoblasts were used as a model system because they havebeen used extensively in in vitro for studies of bone celldifferentiation. These cells undergo a predictable, temporal expressionof biochemical and gene markers of the osteoblast phenotype over a threeto four week period in culture (Aronow 1990, Harris 1994). Lian et al.have described three phases of osteoblast growth and differentiation invitro (Breen 1994). The initial phase (days 1-6) involves active cellproliferation and increases in collagen type I gene expression. Matrixmaturation occurs over the second week in culture and was accompanied byincreased alkaline phosphatase mRNA expression and enzyme activity. Thefinal phase involved cell aggregation into nodules with subsequentmineralization. This period included increased osteocalcin andosteopontin gene expression and protein synthesis. This standardsequence of osteoblast differentiation served as the reference by whichexperimental substrates were evaluated.

In the proposed study, attachment, morphology, proliferation,biochemical markers (alkaline phosphatase and osteocalcin levels) andgene expression (see below for details) for calvarial osteoblasts werequantified after 7, 14, 21, and 28 days in culture. Biochemical assaysand Northern analysis were performed using standard techniques such asthose set forth below.

Methods

Rat Osteoblast Isolation

Primary rat calvarial osteoblasts (RCOB) were isolated from post-natalsix day old rat pups. The crania were dissected using sterile techniqueunder the tissue culture flow hood. Parietal and frontal bonds weredissected free from the sutures and subjected to collagenase digestion(4×20 min; typeI: type II=6:1) (Boden, 1996). The specific activity ofcollagenase I and II (Worthington Enzymes, Freehold, N.J.) was 42.5IU/ml, 88.25 IU/ml in the first digestion and 170 IU/ml, 353 IU/ml forthe remaining two digestions (Boden, supra 1996). Cells from the secondand third digestions were pooled to form an osteoblast rich suspension.These cells were rinsed, pelleted and plated in MEM (Gibco) with 10% FBS(Hyclone) at a density of 6,510 cells per cm²(Lian, 1990). Afterconfluence, the media was switched from MEM to a mineral rich BGJbmedia, to which 10% FBS, 50 mg/ml ascorbic acid, and 10 mM Beta GlycerolPhosphate are added (Lian 1990). For sub-cultivated experiments, primarycells were expanded in T-75 flasks with MEM and 10% FBS. After reaching(about 80%) confluence, cells were sub-cultivated with 2.5% trypsin/EDTAand plated at 20,000 per cm² in MEM+10% FBS (Lian, supra 1990). At day7, 50 mg/ml ascorbic acid was added to induce collagen I synthesis(Boden, supra 1996). At day 14, the media was switched to BGJb+10% FBS,and 10 mM Beta Glycerol Phosphate.

Peptides

Peptides from Table I were synthesized as described above. Each peptidewas coupled to a substrate at a concentration of 0.22M.

Competitive Binding Assays

Experimental and control peptides are added to a suspension of 60,000RCOB cells in serum free media, at a concentration of 0.05, 0.1 and 0.2mM. Cells were incubated with soluble peptide for 45 minutes in ahumidified 5% CO₂, 37° C. environment prior plating ontopeptide-immobilized substrates or the appropriate ECM protein. Allplated cells were maintained in F12 media with no serum. One or twohours after the time of initial plating, a cell count was completed foreach of at least three wells. Cell counting was performed by rinsingseveral times with DMEM, and using the MTT assay (see below) or byfixing with formalin and performing and performing counts in tendifferent high powered microscopic fields on each individual substrate.

Cell Counting with MTT Assay

Standard curves were prepared by plating rat calvarial osteoblasts atdensities of 100,000, 50,000, 25,000, 10,000 and 5,000 cells/well. Cellswere incubated in serum-free Dulbecco's Modified Eagle Media, i.e. DMEM(Gibco) for two hours. Then3-[4.5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, i.e. MTT(Sigma) in media was added to a final concentration of 0.5 mg.ml. Theplates were placed back in the incubator for three hours. Each well wasthen rinsed with Hanks' Balanced Salt and then 1 ml of 10% SodiumDodecyl Sulfate, i.e. SDS (Sigma) was added. Cells were covered inaluminum foil to protect it from light and left at room temperature for12 hours. The SDS solution was then removed, placed into cuvettes, andexamined in a Beckman DU-65 spectrophotometer at a wavelength of 570 nm.

For testing peptide-coated substrates, cells were plated at 50,000 cellsper well plates. Attachment was evaluated at 20 minutes, 1 hour, 3 hoursand 24 hours. At the conclusion of each time oint unattached cells wereremoved by rinsing three times with HBSS. MTT in serum-free media wasadded at a concentration of 0.5 mg/ml to perform cell counting andincubator for 3 hours to allow the cells to process the MTT. The plateswere then removed and each well given a single HBSS rinse followed byaddition of 1 ml of 10% SDS for cell lysis. After 12 hours the SDSsolution was removed, placed into cuvettes, and examined in a BeckmanDU-65 spectrophotometer at a wavelength of 570 nm.

Cell Morphology With Scanning Electron Microscopy

Substrates with cells were treated with 2% paraformaldehyde+1%gluteralehyde for one hour. Then they were rinsed in 1M PBS and placedin 50%, 70%, 90% and 100% (twice) ethanol for ten minutes each todehydrate them. The substrates were then immersed in 1:1ethanol:hexamethyldisilazane (HMDS) for 30 minutes, and finally weretreated with 100% HMDS for thirty minutes and allowed to dry. Sampleswere fixed onto SEM mounts using a conductive graphite adhesive andsputtered with gold. SEM was performed with a Hitachi S2700 scope.

Cell Proliferation Assay (³H-Thymidine Incorporation)

Primary rat calvarial osteoblasts were plated on substrates in 6-wellplates at a density of 20,000 cells/cm², cultured in MEM media(Gibco)+10%FBS (Hyclone) for 2 days, rinsed with buffered saline, andswitched to thymidine-free MEM (Gibco)+0.2% BSA for 1 day (Kim, 1997).On day 4, experimental groups were exposed methyl-³H-thymidine (1uCi/mmol; DuPont New England Nuclear, Boston, Mass., USA) added 4 hoursprior to harvest at 96 hours. Cells were harvested and specificradioactivity (cpm) measured using a scintillation counter. Briefly,cells were washed 3 times with ice-cold PBS to remove excess label,trypsinized, spun down into a pellet, and lysed with 120 ul phosphatebuffered 1% nonidet P-40 (Sigma). One hundred microliters of each samplewere mixed with 800 ul 0.2% BSA, 100 ul 75% trichloroacetic acid (TCA),and centrifuged. Supernatant was removed and the pellet was centrifugedagain with 1 ml 7.5% TCA. The pellet was then solubilized in 900 ml 0.1N NaOH at 37° C. overnight and neutralized with 100 ul 1N Hcl. Countswere normalized with DNA and expressed as cpm/ug DNA. All data wasnormalized using total DNA. A fluorometric DNA assay (Arronow, 1990) wasperformed on the remaining 20 ul aliquots of cell lysate using a TKO 100mini-fluorometer (Hoefer, San Francisco, Calif., USA) to normalize cellcounts. Samples were incubated with benzimidazole (Hoechst 33258;Pharmacia Biotech, Piscataway, N.J.) and fluorescence quantified. DNAcontent was obtained using a calf thymus DNA (Pharmacia Biotech)standard curve.

Alkaline Phosphatase Activity

Alkaline phosphatase (AP) activity of cell lysates was determined by anestablished enzymatic conversion assay using p-nitrophenol phosphate asa substrate (Spiess). The enzyme activity was expressed as nanomoles ofp-nitrophenol produced per minute per milligram of protein (nmol/min/mgprotein). The protein content was determined using the Biorad proteinassay kit (Biorad, Hercules, Calif.) using BSA as the standard.

Alkaline Phosphatase Staining

Osteoblasts were fixed in buffered 2% paraformaldehyde for 24 hours.Before staining, cells were rinsed in distilled water. Alkalinephosphatase staining was visualized by incubating the cells for 30 minin 0.1 M Tris HCL pH 8.5 containing 0.4 mg/ml naphthol AS-MX phosphate+1mg/ml Fast Blue BB salt. Cells were then rinsed in 1 M PBS and preservedin PBS glycerol. The intensity of osteoblasts stained with alkalinephosphatase was qualitatively assessed by counting the number ofosteoblasts per 10× field.

Osteocalcin RIA

Osteocalcin levels were assessed after removing aliquots of conditionedmedia from cell cultures of experimental groups using a radioimmunoassay(RIA) for rat osteocalcin (rat osteocalcin kit, BTI, Stoughton, Mass.)according to a previously described method (Gundberg 1984). Purified ratosteocalcin, goat anti-rat osteocalcin antibody, normal goat nonimmuneserum, donkey anti-goat 2nd antibody, RIA buffer and [I-125] ratosteocalcin were used as reagents for RIA as provided by BTI. Thestandards and the samples were incubated overnight with a known quantityof goat anti-rat osteocalcin antibody followed by another incubationwith [I-125] rat osteocalcin (approx. 20,000 cpm). The tubes wereincubated (2 hours) with donkey anti-goat 2nd antibody, centrifuged, andpellets counted (cpm) using a gamma counter. A standard curve wasgenerated and sample concentration of osteocalcin (ng/tube) obtained.

Extracellular Matrix Protein and Integrin Gene Expression by NorthernAnalysis

During the course of bone development and metabolism, a variety ofosteoblast growth and differentiation factors are known to be expressedin vitro (Ibaraki, 1992) and in vivo (Jingushi, 1991, Sandberg, 1993).Integrin gene expression is also modulated.

1. RNA extraction

RNAzolTM (Tel-Test, Friendswood, Tex.) reagent was added to the cellcultures removed of media and then shaken gently until a viscous, opaqueliquid was seen. The contents were transferred to ice cold tubes towhich chloroform was added and vortexed. After centrifugation for 10min. at 10,000 rpm, the top aqueous phase was re-extracted with freshRNAzol and chloroform. After a series of re-extraction andcentrifugations, the cell pellet was washed in 70% ethanol andresuspended in 50 ul TE buffer. The concentrations and purity of the RNAis measured with a spectrophotometer using the ratio of A²⁶⁰ and A²⁸⁰.

2. RNA gel

RNA (15-20 ug) from specific experimental groups was separated on thebasis of size with a denaturing 1.2% agarose (Formaldehyde) gelelectrophoresis. All RNA gels were run for 3 hours at 100 V andphotographed using an ultraviolet light source. A TurboBlot % kit wasused to transfer the RNA from the gel to the nylon (blotting) membrane.The RNA was then cross linked and baked on permanently onto the membrane(under a UV lamp and baked at 120° C. for 15 minutes).

3. Hybridization & Detection

cDNA probes for rat alkaline phosphatase and rat osteocalcin were kindlyprovided by Dr. J. Lian (University of Massachusetts, Worcester, Mass.).The cDNA probes for α5 and β1 integrins were provided by Dr. E.Ruoslahti (Cancer Institute, La Jolla, Calif.). The cDNA probe for bonesialoprotein was provided by Dr. J. Sodek (University of Toronto,Toronto, Canada), while the cDNA probe for collagen was provided by Dr.B. Kream (University of Connecticut, New London, Conn.). The cDNA probesfor GAPHDH, beta-actin, human osteopontin, and human osteonectin wereobtained from the American Type Culture Collection (ATCC).

The membranes were treated with various buffers (5×SSPE, 50% formamide,2% SDS and 10×Dengardt's solution). The hybridized probes wereradioimmunodetected through the use of ³²P. The membranes were thenreacted with the cDNA probes and hybridized with the probes at 68° C.overnight and washed through a series of (2×SCC & 0.1% SDS) solutions.After the washing steps, the membrane were exposed with x-ray film. Themount of RNA was quantified through comparison with the known amount ofRNA transcript that was loaded in the lane on the gel. The size of theRNA molecule was calculated by measuring the distance migrated andcomparing it to the standard. All mRNA hybridization experiments wereperformed twice with each cDNA probe. All cDNA was normalized to GAPDH.

Results

Cell Attachment

RCOBs in DMEM with 10% fetal bovine serum were plated at 25,000 persquare centimeter. Visual analysis revealed higher levels of attachmentat 20 minutes on the RGDC treated substrates. This was quantitativelyconfirmed using an MTT assay which showed that at 20 minutes there was100% greater attachment to the RGDC surface compared with gold and RGDtreated surfaces. Similar to the gold surface RCOBs showed much greaterattachment when cultured on RGDC-FEP modified surfaces than onunmodified FEP.

Alkaline phosphatase Activity

Alkaline phosphatase activity revealed that RGDC coupled surfacesproduced the highest levels of this enzyme. CGRARADSP (control peptide)and plain gold substrates produced values which were not significantlydifferent from one another. The results are shown in FIG.

Osteocalcin mRNA Assay

At nine days Osteocalcin mRNA was heavily expressed on RGDC-gold coatedsurfaces but was not observed on Gold and CG-gold coated surfaces. Afterfourteen days RGDC still showed higher levels than the others, and atnineteen days all substrates were virtually identical. Additionally itwas found that Osteocalcin mRNA expression was induced earlier in cellswhich were grown on the prosthetic implants of the invention having apeptide coated gold surface than on the polymeric prosthetic implants(FEP) coated with the identical peptide. Osteocalcin is not expressed at9 days when cells are grown on the peptide coated polymer surface. Thisfinding suggest that the gold surface is also important to theregulation of bone morphogenesis.

Osteocalcin Protein Synthesis

It is also important to determine how immobilized proteins influenceprotein synthesis. Osteocalcin was evaluated since it is a marker ofbone cell differentiation and because radioimmunoassays (RIA) arecommercially available (Arono, 1990; Gundberg, 1984; Ibaraki. 1992).

After 14 days in culture, RGD-FEP coupled membranes inducedsignificantly higher levels of osteocalcin synthesis compared with allother groups. The unique ability of RGD-FEP coated substrates to enhanceosteocalcin synthesis is consistent with increased RCOB mRNA expressionseen at day 14. The RGE is closest to RGD in stimulating osteocalcinsynthesis. RAD peptide was similar to OH and TCP.

ECM and Integrin Gene Expression for Sub-Cultivated RCOBs

Evaluation of matrix protein gene expression provides a quantitativemethod of assessing cell differentiation in a temporal fashion. Thenormal pattern of RCOB gene expression has been reported for primary andsubcultured cells (Aronow, 1990; Breen, 1994; Lynch, 1995 and others).It has been demonstrated that subcultured RCOB display a “right-shifted”pattern of gene expression compared to primary cells.

Alkaline Phosphatase

On day 9 Alkaline Phosphatase gene expression was observed on allsubstrates at minimal levels but was significantly higher on RGDC coatedgold surfaces. No change over the time period studied in AlkalinePhosphatase levels was observed in the cells cultured on FEP surfaces.

Bone Sialoprotein

Similar to Alkaline Phosphatase gene expression, bone sialoprotein geneexpression was much higher on RGDC coated gold surfaces than on goldsurfaces alone or gold surfaces coated with a control peptide. Bonesialoprotein gene expression was not observed in cells cultured on FEPsurfaces.

β₁ integrin

β₁integrin gene expression was observed on all substrates at minimallevels but was significantly higher on RGDC coated gold surfaces. By the14th day of culture, the mRNA signal detected from cells cultured onRGDC coated gold surfaces had shifted from one band to two bands. Thisshift to two bands was not detected in RNA isolated from cells culturedon any of the other surfaces.

α₅ integrin

α₅ integrin gene expression was observed in cells cultured on RGDCcoated gold surfaces but was not detected in cells cultured on any othersurfaces. Similar to β₁ integrin, the expression pattern of α₅ integrinwas observed to shift on day 14 from a single band to a double band.

Example 3 Peptide Modified Surfaces Support Focal Adhesion Formation

The cytoplasmic domains of integrins are relatively short (approximately50 amino acids), but are sufficiently long enough to interact withcytoskeletal proteins in focal contacts (or focal adhesions or adhesionplaques). Focal adhesions are connected to the nucleus via microspikesor bundles of actin filaments. Several experiments provide strongevidence for these connections between the exterior and interior of thecell. In fluorescence photobleaching, integrins were fluorescentlylabeled, then overexposed to form a bleached spot. This bleached areadid not move, showing the restricted mobility of integrins in focalcontacts (Duband, 1986). Solowska (1989) showed that expression of amutant form of avian integrin beta 1 subunit lacking the cytoplasmicdomain produces hybrid heterodimers which, while efficiently exported tothe cell surface and still capable of binding fibronectin, do notlocalize efficiently in focal adhesions. This further implicates thecytoplasmic domain of the beta 1 subunit in interactions required forcytoskeletal organization.

The cytoskeletal proteins present in focal adhesions are well-defined:vinculin, talin, and alpha actinin serve as links between integrins andthe bundles of actin filaments (stress fibers) of the cytoskeleton.Evidence in the literature suggests that focal adhesions are requiredfor signal transduction from the ECM to the nucleus of the cell. Uponintegrin-mediated adhesion to ECM proteins, focal adhesion kinase (FAK),a tyrosine kinase, becomes phosphorylated (Schneider, 1994). Activationof FAK is believed to initiate a signaling pathway to the nucleus,resulting in changes in gene expression.

Both fibronectin and type I collagen are present in the extracellularmatrix. We tested our monolayer surface of active peptides to determinewather it can stimulate focal adhesion formation in the absence of serumin a similar manner to the interaction between the cell and the relatedparent protein of the peptide sequence. In order to evaluate theinfluence of active portions of these parent proteins on osteoblast cellresponse in short time frames we modified gold coated coverslips withthe fibronectin related peptide: RGDC or the collagen related peptide:DGEAGC, and evaluated the ability of these surfaces to support focaladhesion formation at two time points, 3 and 24 hours, under serum freeconditions.

Methods

Fibronectin/RGDC Study

Experimental groups included RGDC, RADC, fibronectin adsorbed to gold,plain gold, and plain glass surfaces. Gold substrates were manufacturedby evaporating 80 angstroms of titanium to 12 mm glass coverslips(Fisher), followed by a 500 angstrom layer of gold. To couple cysteineterminated peptides to the gold substrates, a 0.22 mM solution of thedesired peptide was solubilized in a 1:1 mixture of distilled water andethanol. These substrates were then incubated overnight. Plain goldcontrols were exposed to ethanol and distilled water as well.Fibronectin substrates were produced by incubating gold coverslips with10 μg/ml of fibronectin (Collaborative Biomedical Products, Bedford,Mass.) for 60 minutes, followed by 10 mg/ml bovine serum albumin (BSA)(Sigma, St. Louis, Mo.) for 30 minutes to cover any non-specific bindingsites. All coverslips were then washed three times in HBSS to remove anynon-adsorbed protein (Puleo, 1991). Primary rat calvarial osteoblastswere isolated according to protocol and seeded for periods of 3 or 24hours in serum free or serum conditions. At each time point cells wererinsed in warm PBS, fixed in 3.7% paraformaldehyde for 30 minutes, andrinsed several times with HBSS. Vinculin and actin were labeled via thefollowing protocol: nonspecific sites were blocked in 5% BSA for 30minutes, cells were then permeabilized with 0.2% Triton X-100 (Fisher)for 10 minutes, incubated in a 1:50 mouse anti-human vinculin antibodysolution (Sigma St. Louis, Mo.), blocked for 30 minutes, and incubatedwith a anti-mouse rhodamine secondary antibody (1:50) and FITCconjugated phalloidin (Molecular Probes).

Type I Collagen/DGEAGC

Experimental groups included DGEAGC and rat tail type I collagenadsorbed to gold, plain gold, and plain glass surfaces. Gold substrateswere manufactured by evaporating 80 angstroms of titanium to 12 mm glasscoverslips (Fisher), followed by a 500 angstrom layer of gold. To couplecysteine terminated peptides to the gold substrates, a 0.22 mM solutionof the desired peptide was solubilized in a 1:1 mixture of distilledwater and ethanol. These substrates were then incubated overnight. Plaingold controls were exposed to ethanol and distilled water as well. TypeI collagen substrates were produced by incubating gold coverslips with10 μg/ml of collagen (Collaborative Biomedical Products, Bedford, Mass.)for 60 minutes, followed by 10 mg/ml bovine serum albumin (BSA) (Sigma,St. Louis, Mo.) for 30 minutes to cover any non-specific binding sites.All coverslips were then washed three times in HBSS to remove anynon-adsorbed protein (Puleo, 1991). Primary rat calvarial osteoblastswere isolated according to protocol and seeded for periods of 3 or 24hours in serum free or serum conditions. At each time point cells wererinsed in warm PBS, fixed in 3.7% paraformaldehyde for 30 minutes, andrinsed several times with HBSS. Vinculin and actin were labeled via thefollowing protocol: nonspecific sites were blocked in 5% BSA for 30minutes, cells were then permeabilized with 0.2% Triton X-100 (Fisher)for 10 minutes, incubated in a 1:50 mouse anti-human vinculin antibodysolution (Sigma St. Louis, Mo.), blocked for 30 minutes, and incubatedwith an anti-mouse rhodamine secondary antibody (1:50) and FITCconjugated phalloidin (Molecular Probes).

Results

At three hours, vinculin staining revealed the ability of RGDC peptidemodified surfaces to support focal adhesion formation in the absence ofserum. Fibronectin coated surfaces also supported focal adhesionformation. Cells on both surfaces tended to have vinculin staininglocated at the cell periphery in the form of distinct plaques at thecell tips. No vinculin staining was observed on cells plated on RADC,glass or plain gold.

At three hours, vinculin staining revealed the ability of DGEAGC peptidemodified surfaces to support focal adhesion formation in the absence ofserum. Collagen coated surfaces also supported focal adhesion formation.Cells on both surfaces tended to have the brightest vinculin staininglocated at the cell periphery in the form of either distinct plaques orgroups of strands at the cell tips. No vinculin staining was observed oncells plated on glass or plain gold.

Example 4 Evaluation of the Effect of Immobilized Peptides on OsteoblastDifferentiation

We have shown above that in vitro, peptide modified surfaces caninfluence short and long term cell responses like attachment, shape andfunction. We also conducted a study to evaluate the amount of boneformed in response to gold coated titanium rods modified with thepeptide sequence Arg-Gly-Asp-Cys (RGDC). Titanium rods coated with gold,FEP rods and uncoated titanium rods were implanted bilaterally into thedistal medial femoral condyle of adult rats and evaluated at 2, 4, 8,and 24 weeks post-implantation. The experiments are discussed below.

In vivo

Modification and Characterization of Peptide-grafted FEP Rods andTitanium

Titanium rods were generously donated by Osteonics Corporation (NJ,USA). Rods were cleaned according to ASTM standards before coating themwith a 500 layer of gold using an electron beam evaporator. Rods wereimmersed in a 0.22 M solution (1:1 ethanol:water) of RGDC (AmericanPeptide Company, Sunnyvale, Calif.) overnight at room temperature andstored in sterile PBS, using the techniques described above, until thetime of surgery. FEP rods were coupled with peptides using thetechniques described above. Un-coated titanium rods are used as acontrol. Briefly, the materials were initially cleaned in a radiofrequency glow discharge chamber using a flow-through system with anArgon atmosphere. The alloys were immediately transferred to a nitricacid bath for 30 min in order to passivate the surface according to ASTMstandards (Puelo 1994). The samples were transferred to a goldevaporation chamber and reacted with peptides as described above.Characterization of gold coated titanium materials, FEP and titaniummaterials were performed as described above.

Peptide-coated Materials and uncoated Titanium Implanted in Rat FemurSites

Quantitative histomorphometric analysis and pull-out biomechanicaltesting was conducted at 2 and 4 weeks on implants inserted bilaterallyinto the femoral canal of 20 adult Sprague Dawley rats. Parametersevaluated included the area and thickness of new bone formed around theimplants, the percent of the implant covered by new bone, and theinterfacial shear strength at the bone/implant interface. The distal ratfemur provides a well-studied site for bone material interactions andoffers a sufficient bony area to implant small specimens. Adult SpragueDawley rats weighed an average of 415±12 g at the time of surgery. Therats were anesthetized using a 0.5 ml intraperitoneal injection ofNembutal and 0.1 ml of Cefazolin was injected intramuscularly at thesurgical site. Reaming of the distal end of the femoral canal was donefirst by inserting an 18 gauge needle down the femoral shaft, followedby irrigation of the femur with sterile saline, reaming with a 1.5 mmdrill bit using a hand held drill to prevent thermal necrosis,irrigation, reaming with a 16 gauge needle, irrigation, and finalreaming of the outer cortex with a 14 gauge needle. The rod was thenpress fit into place with the outermost end below the condylar surface,in each case. RGDC coated rods were placed at random with one controlrod and one experimental rod being placed bilaterally in each animal.Lateral and anterior-posterior X-rays were taken postoperatively toassess rod position. The fascia and skin are closed in standard fashionusing 5-1 vicryl bioresorbable sutures.

Histological evaluation

After mechanical testing, femurs were removed from the dental plasterand stored in phosphorous buffered saline for 24 hours until fixation in3.7% paraformaldehyde for 48 hours at room temperature. Decalcificationwas performed according to a method described by Frank, et al, (1993).Briefly, bones were allowed to demineralize over the course of 2 weeksin 15% formic acid solution at 4 ûC. Bones were rinsed and permbealizedin 6.8% sucrose/PBS solution overnight. Dehydration of bones wasconducted as follows: 20 minutes per ethanol concentration: 70, 80, 90,95%. Bones were sectioned from the growth plate at 2, 5, 8, 12, and 15mm and embedded in Historesin (Leica, Germany) for histologicalanalysis. Briefly, bones were infiltrated for 48 hours at 4 ûC and thenembedded overnight. 5 μm sections were made. Specimens were stainedusing Hematoxylin and Eosin and GomoriÕs trichrome stains. Quantitativehistomorphometrical analysis was conducted on bone cross sectionssectioned at 5 mm using IP Lab Software. Images of bone cross sectionswere captured by microscope and imported into a computer via a CCDcamera. Parameters measured by two independent observers included theperimeter of new bone formed, the area of new bone, the perimeteravailabe for new bone formation and the diameter of the hole where theimplant was. Some sections were not analyzed due to histologicalsectioning tears. Also, if the amount of new bone formed around theimplant was not clear (e.g. implant abutting cortex) that portion of thecross section was not included for analysis.

Biomechanical Pull-out Testing

The biomechanical pull-out strength between the bone/RGDC and bone/Auwas measured using the widely imployed pull-out test (Chae et al in 1992and Tisdel et al in 1994, Branemark & Berzin). All testing was performedin a blinded fashion.

Animals were sacrificed at 2 and 4 weeks postoperatively. Animals werefirst anesthetized with a 0.5 ml intraperitoneal injection of Nembutaland then sacrificed by a 0.5 ml intracardial injection of Nembutal.Mechanical testing of all femurs was conducted the same day assacrifice. Immediately after explantation, femurs were cleaned of allsoft tissue, x-rayed and prepared for mechanical testing or histologicalevaluation. A total of 23 animals were evaluated. Three animals wereexcluded, one because of death during surgery, and two because ofpathologic fractures. For mechanical testing at 2 weeks, 7 animals wereassessed while 8 animals were assessed at 4 weeks. Histologicalevaluations were carried out on 8 femurs used for mechanical testing ateach time point. An alignment jig was designed in order to insure a puretensile force was applied to the rod. Dental plaster was used to holdthe proximal portion of the femur in place during testing. Modifiedneedle-drivers gripped the end of the implant as it was pulled from thebone at a crosshead speed of 5 m/min. The force required to break theinterface was recorded and the portion of the implant estimated to becontact bone was also recorded.

Results

The in vivo studies involving implantation of FEP coated and uncoatedmaterials indicate that implants coated with a bioactive molecule suchas RGDC have accelerated or enhanced bone ingrowth. Briefly, the peptidecoated implants demonstrated a significantly greater percentage of theirsurface perimeter covered with bone. Additionally the biomechanicalpull-out strength was significantly greater for the peptide coatedimplants versus the uncoated implants.

By 4 weeks the average pull-out force of peptide modified rods was 38%greater than gold control rods although this difference was notstatistically significant (Table 1). Furthermore, at 4 weeks there wassignificantly (P<0.01) more new bone area formed around RGDC implantsand the thickness of this new bone formed around RGDC implants differedsignificantly (P<0.01) from Au controls at both 2 weeks (26.2microns±1.9 vs. 20.5 microns±2.9) and 4 weeks (32.7 microns±4.6 vs. 22.6microns±4.0)

Biomechanical

No statistical differences were found between peptide modified and goldcontrol rods for the interfacial shear strength at 2 and 4 weeksrespectively. It should be noted however, that the mean of the peptidemodified group at 4 weeks was 38% higher than the control group (Table1).

TABLE 1 Interfacial Shear Strength (MPa) 2 weeks Postimplantation 4weeks Postimplantation Gold coated rods 0.17 ± 0.09 0.13 ± 0.06 RGDCmodified 0.16 ± 0.06 0.18 ± 0.07 rods

Histology

Although there were not a significant differences in the pull-out forcesbetween groups, there were significant differences in the amount of bone(thickness and area) formed around the implants at two and four weeks.There were no differences in the percent of the implant cross sectioncovered by bone (76±14%, 74±5%) for the RGDC and Au groups respectively.At four weeks more of the implant was covered by bone but the percent ofthe implant cross section covered by bone for the RGDC and Au groups didnot differ significantly (92±4% vs. 90±7%). The area of new bone formedaround RGDC implants was not significantly more compared to Au controlsat 2 weeks (0.108 μm²±0.026 vs. 0.082 μm²±0.017), but by 4 weeks therewas a significantly (P<0.01) more area of new bone formed around RGDCimplants (0.16 μm²±0.016 vs. 0.108 μm²±0.023). The thickness of new boneformed around RGDC implants differed significantly (P<0.01) from Aucontrols at both 2 weeks (26.2 μm±1.9 vs. 20.5 μm±2.9) and 4 weeks (32.7μm±4.6 vs. 22.6 μm±4.0).

Example 5 Peptides Act Synergistically to Increase Bone CellResponsiveness

The response of bone cells to peptide combinations showing synergy orhigh individual levels of activity is evaluated in vitro and in vivousing methods described above with combinations of peptides rather thana single peptide. Two non-adjacent peptide sequences from fibronectin,including RGD and PHSRN, a so-called synergy sequence, exhibitsynergistic behavior (Aota 1994; Akiyama, 1995). These experiments canbe used to identify, track and quantify different peptides on the samemembrane.

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Each of the foregoing patents, patent applications and references isherein incorporated by reference in its entirety. Having described thepresently preferred embodiments in accordance with the presentinvention, it is believed that other modifications, variations andchanges will be suggested to those skilled in the art in view of theteachings set forth herein. It is, therefore, to be understood that allsuch variations, modifications, and changes are believed to fall withinthe scope of the present invention as defined by the appended claims.

What I claim is:
 1. A prosthetic device, comprising: a shaped substratehaving a substrate surface, for implantation in a mammal; a layer ofgold attached to the substrate surface and defining a tissue contactingsurface; wherein the gold layer has a thickness of about 10 to 1000Angstroms and, a bioactive peptide bound to the gold layer.
 2. Thedevice as in claim 1, wherein the bioactive peptide is selected from thegroup consisting of a cell modulating peptide, a chemotactic peptide,anticoagulant peptide, antithrombotic peptide, an anti-tumor peptide, ananti-infectious peptide, a growth potentiating peptide, and ananti-inflammatory peptide.
 3. The device as in claim 2, wherein the cellmodulating peptide is selected from the group consisting of ananti-integrin antibody fragment, a cadherin binding peptide, and anintegrin binding peptide.
 4. The device as in claim 3, wherein the cellmodulating peptide is an integrin binding peptide which is selected fromthe group consisting of RGDC, RGEC, RGDT, DGEA, DGEAGC, EPRGDNYR, RGDS,EILDV, REDV, YIGSR, SIKVAV, RGD, RGDV, HRNRKGV, KKGHV,XPQPNPSPASPVVVGGGASLPEFXY, and ASPVVVGGGASLPEFX.
 5. A prosthetic device,comprising: a shaped substrate having a substrate surface, forimplantation in a mammal; a layer of gold attached to the substratesurface and defining a tissue contacting surface; and, a bioactivepeptide bound to the gold layer wherein the bioactive peptide is achemotactic peptide and, wherein the chemotactic peptide is selectedfrom the group consisting of functionally active fragments of collagen,fibronectin, laminin, and proteoglycan.
 6. A prosthetic device,comprising: a shaped substrate having a substrate surface, forimplantation in a mammal; a layer of gold attached to the substratesurface and defining a tissue contacting surface; and, a bioactivepeptide bound to the gold layer, wherein the bioactive peptide is ananti-tumor peptide and, wherein the anti-tumor peptide is selected fromthe group consisting of functionally active fragments of proteinanti-tumor molecules.
 7. The device as in claim 2, wherein theanti-infectious peptide is selected from the group consisting offunctionally active fragments of the protein anti-infectious molecules.8. A prosthetic device, comprising: a shaped substrate having asubstrate surface, for implantation in a mammal; a layer of goldattached to the substrate surface and defining a tissue contactingsurface; and, a bioactive peptide bound to the gold layer, wherein thebioactive peptide is a growth potentiating peptide and, wherein thegrowth potentiating peptide is selected from the group consisting offunctionally active fragments of PDGF, EGF, FGF, TGF, NGF, CNTF, GDNF,and type I collagen peptides.
 9. The device as in claim 2, wherein theanti-inflammatory peptide is selected from the group consisting offunctionally active fragments of anti-inflammatory molecules.
 10. Thedevice as in claim 1, wherein the layer of gold is attached to thesubstrate surface via attachment to a layer of titanium intermediate thegold layer and the substrate surface.
 11. The device as in claim 1,wherein the shaped substrate is selected from the group consisting of apolymer, a metal, a plastic, a fabric, a ceramic, a biological material,and a composite of two or more materials.
 12. A prosthetic device,comprising: a shaped substrate having a substrate surface, forimplantation in a mammal; a layer of gold attached to the substratesurface and defining a tissue contacting surface; and, a bioactivepeptide bound to the gold layer, wherein the bioactive peptide forms alayer about 1 to 500 Angstroms in thickness.
 13. The device as in claim1, wherein at least two bioactive peptides are bound to the surface. 14.A prosthetic device, comprising: a shaped substrate having a substratesurface, for implantation in a mammal; a layer of gold attached to thesubstrate surface and defining a tissue contacting surface; and, abioactive peptide bound to the gold layer, wherein the bioactivemolecule is bound to the gold by a gold-sulfide bond.
 15. A prostheticdevice, comprising: a shaped substrate formed of a textured materialhaving a substrate surface with first projections and firstindentations; and a layer of gold attached to the substrate surface ofthe textured material, wherein the layer of gold creates a gold surfacethat has second projections said second indentations corresponding tosaid first projections and indentations.
 16. The device as in claim 15,wherein the layer of gold has an approximately uniform thickness acrossthe substrate of the textured material.
 17. The device as in claim 15,wherein the textured material is a polymer.
 18. The device as in claim15, wherein the gold layer has a thickness of about 10 to 1000Angstroms.
 19. The device as in claim 15, further comprising a layer ofbioactive peptide attached to the gold surface through a gold-sulfidebond.
 20. A prosthetic device, comprising: a shaped substrate having asubstrate surface; a layer of gold attached to the substrate surface;and an RGDC peptide attached to the gold layer through a gold-sulfidebond, wherein the RGDC peptide forms a layer about 1 to 500 Angstroms inthickness.
 21. The device as in claim 20, wherein the layer of gold isattached to the substrate surface via attachment to a layer of titaniumintermediate the gold layer and the substrate surface.
 22. The device asin claim 20, wherein the shaped substrate is selected from the groupconsisting of a polymer, a metal, a plastic, a fabric, a ceramic, abiological material, and a composite of two or more materials.
 23. Aprosthetic device, comprising: a shaped substrate having a substratesurface; a layer of gold attached to the substrate surface; and an RGDCpeptide attached to the gold layer through a gold-sulfide bond, whereinthe gold layer has a thickness of about 10 to 1000 Angstroms.
 24. Aprosthetic device, comprising: a shaped substrate having a substratesurface; a layer of gold attached to the substrate surface; and an RGDCpeptide attached to the gold layer through a gold-sulfide bond, whereinthe surface of the prosthetic device is formed of a porous material andwherein the layer of gold creates a gold surface that has projectionsand indentations and wherein the layer of gold has an approximatelyuniform thickness across the surface of the porous material.
 25. Aprosthetic device, comprising: a shaped substrate having a substratesurface, for implantation in a mammal; a layer of gold attached to thesubstrate surface and defining a tissue contacting surface; and, abioactive molecule bound to the gold layer, wherein the bioactivemolecule is selected from the group consisting of a cell modulatingmolecule, a chemotactic molecule, anticoagulant molecule, antithromboticmolecule, an anti-tumor molecule, an anti-infectious molecule, a growthpotentiating molecule, and an anti-inflammatory molecule.
 26. The deviceas in claim 25, wherein the cell modulating molecule is selected fromthe group consisting of an antibody, a bone morphogenic protein, anintegrin binding protein, and a cadherin binding protein.
 27. The deviceas in claim 26, wherein the cell modulating molecule is a bonemorphogenic protein.
 28. The device as in claim 25, wherein thechemotactic molecule is selected from the group consisting of collagen,fibronectin, laminin, and proetoglycan.
 29. The device as in claim 25,wherein the anti-tumor molecule is selected from the group consisting ofmethotrexate, adriamycin, cyclophosphamide, and taxol.
 30. The device asin claim 25, wherein the anti-infectious molecule is selected from thegroup consisting of antibiotics such as penicillin.
 31. The device as inclaim 25, wherein the growth potentiating molecule is selected from thegroup consisting of PDGF, EGF, FGF, TGF, NGF, CNTF, and GDNF.
 32. Thedevice as in claim 25, wherein the anti-inflammatory molecule isselected from the group consisting of steroidal and non-steroidalcompounds.
 33. The device as in claim 25, wherein the layer of gold isattached to the substrate surface via attachment to a layer of titaniumintermediate the gold layer and the substrate surface.
 34. The device asin claim 25, wherein the shaped substrate is selected from the groupconsisting of a polymer, a metal, a plastic, a fabric, a ceramic, abiological material, and a composite of two or more materials.
 35. Aprosthetic device, comprising: a shaped substrate having a substratesurface, for implantation in a mammal; a layer of gold attached to thesubstrate surface and defining a tissue contacting surface; and, abioactive molecule bound to the gold layer, wherein the gold layer has athickness of about 10 to 1000 Angstroms.
 36. A prosthetic device,comprising: a shaped substrate having a substrate surface, forimplantation in a mammal; a layer of gold attached to the substratesurface and defining a tissue contacting surface; and, a bioactivemolecule bound to the gold layer, wherein the bioactive molecule forms alayer about 1 to 500 Angstroms in thickness.
 37. A prosthetic device,comprising: a shaped substrate having a substrate surface, forimplantation in a mammal; a layer of gold attached to the substratesurface and defining a tissue contacting surface; and, a bioactivemolecule bound to the gold layer, wherein the surface of the prostheticdevice is formed of a porous material and wherein the layer of goldcreates a gold surface that has projections and indentations saidcorresponding to the projections and indentations.
 38. The device as inclaim 37, wherein the layer of gold has an approximately uniformthickness across the surface of the porous material.
 39. The device asin claim 37, wherein at least two bioactive peptides are bound to thesurface.